ABSTRACT A central question in cell biology is how spatial information on the micron-length scale is encoded within the cellular cytoplasm. This is essential for maintaining genome integrity during mitosis where the cell-cleavage plane must be precisely positioned at the center of a ~ 10-15 um long cell during cytokinesis. It is now well established that the microtubule cytoskeleton plays a critical role in this process by forming a specialized structure proximal to the site of cell cleavage. This structure, known as the spindle midzone, acts as a micron scale `mark' for the cell center. These micron-scale marks are then `read' by signaling proteins that generate self- organizing activity patterns that further precisely define the site of cell-cleavage. Subsequently this information is transmitted to the cell cortex for the organization of the contractile ring. We now have a near complete parts list of the proteins that regulate these processes. However, the molecular mechanism by which the midzone templates the formation of spatial activity patterns of regulatory proteins within the cytoplasm and the cell cortex is starkly incomplete. Here we will develop in vitro reconstitution assays using microtubules, reconstituted bilayer, singe molecule FRET sensors and DNA origami-based nanotemplates to decipher how spatial organization of signaling activity is achieved on microtubule templates and how these reactions are organized at membrane-microtubule interface. These studies will provide insights into a conserved signaling cascade that is critical for cell division and its mis-regulation in human cancer. Importantly, the innovative methodologies developed here will be widely applicable to dissecting other signaling mechanisms by which spatial activity patterns are encoded and deciphered in the cellular cytoplasm during cell division, growth and development.